The overall goal of this research is to elucidate molecular mechanisms by which Ca2+, cooperation, and protein phosphorylations regulate contraction of mammalian myocardium. The objective of this proposal is to elucidate the roles of myosin binding protein-C (cMyBP-C) in myocardium, with particular emphasis on the regulation of contraction and the effects of cMyBP-C phosphorylation on force and the kinetics of force development. (1) We hypothesize that cMyBP-C modulates contraction by binding to myosin subfragment 2 (S2), thereby physically controlling the availability of cross-bridges to actin. This idea will be tested by assessing the functional effects of interventions designed to disrupt cMyBP-C/S2 interactions in mouse skinned myocardium and using X-ray diffraction and electron microscopy to assess the structural effects of these interventions. (2) We hypothesize that the altered systolic function we have observed in our cMyBP-C null mouse results from accelerated cross-bridge kinetics and stretch activation due to deletion of cMyBP-C. We will test these ideas by measuring the activation dependence of the rate of rise of force and stretch activation in null myocardium and assessing the reversibility of these effects by reconstituting null myocardium with cMyBP-C. (3) We hypothesize that at least some of the positive inotropy induced by (alpha-adrenergic agonists is due to protein kinase A-mediated phosphorylation of cMyBP-C. We will test this idea by assessing the effects of PKA on the force and kinetics of force development (i) in mouse myocardium expressing mutant cardiac Tnl that cannot be phosphorylated by PKA, (ii) in cMyBP-C null myocardium expressing non-phosphorylatable cTnl, and (iii) in myocardium expressing mutants of cMyBP-C in which phosphorylatable serines are replaced with alanines or with aspartates. The idea that the effects of phosphorylation are due to alterations in cross-bridge availability to actin will be assessed by X-ray diffraction. The possibility that the contractile phenotypes of knock-out and transgenic mice are due in part to compensatory mechanisms will be studied both by reconstitution of null myocardium with wild-type and mutant proteins and by conditional expression of null and mutant alleles. Results should provide new information about mechanisms by which contractile state is modulated in healthy myocardium and new insights as to the basis for functional deficits in diseased hearts.